With a mortality rate of 30% and high rates of aerosol transmission, smallpox is among the most deadly potential agents of bioterrorism. Treatment options are limited, and vaccinia virus, the current smallpox vaccine, may induce serious side effects. Studies directed at both vaccinia virus biology and human immunity will refine vaccines and treatments for emerging poxvirus infections. Neutralizing the innate immune response is critical to poxvirus survival. Toll-like receptors (TLRs) are pathogen pattern-recognition molecules that mediate the innate immune response, and vaccinia virus encodes proteins interdict signal transduction and anti-viral activity of TLRs. Our database searches identified a vaccinia virus inhibitor of TLR signaling as structurally similar to the vaccinia virus N1L protein. Although its function was previously unknown, vaccinia virus N1L was a robust determinant of vaccinia virus virulence in two independent in vivo studies. Vaccinia viruses deleted of the N1L gene required 10,000-fold higher titers of virus to cause infections in vivo, suggesting that N1L inhibits innate resistance to infection. Furthermore, N1L is 98% identical to its smallpox virus homolog, suggesting N1L contributes to the virulence of variola (smallpox) virus. Finally, N1L-deleted vaccinia virus had been proposed as a vaccine candidate because of its relative avirulence and immunogenicity. Our data indicate that N1L inhibits signaling to NF-kappaB via TLRs, IL-1 R, and receptors for TNF superfamily members. Since all of these signals proceed via the IKK complex, we first propose to test the hypothesis that N1L inhibits signaling to NF-kB by targeting the IKK complex. Then we will test whether N1L-deficient virus no longer inhibits signaling to NF-kappaB. Next we will determine whether overexpression of the cellular target of N1L during vaccinia virus infection suppresses the N1L phenotype. Finally, we will create recombinant vaccinia virus encoding N1L domain-deletion mutations and test for attenuated pathogenicity and immune responses in mice. This will increase understanding of innate immunity in the pathogenesis of vaccinia virus infections. These studies will advance our long-term goal: to characterize the mechanisms involved in viral subversion of signal transduction that contribute to viral pathogenesis.